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At each sampling, the site pH was measured with a portable pH meter, salinity was determined with a refractometer, and the water, air temperature, and water depth were recorded. The average values and standard error for each of these parameters were calculated over the course of each experiment. Scanning electron microscopy SEM samples were placed on filters and subjected to an ethanol dehydration series, mounted on aluminum stubs with carbon tape, critical point dried, and sputter coated with carbon.
High-throughput amplicon sequencing of the SSU rRNA gene was used to assess community composition and diversity of both steel coupon biofilms and sediments.
The samples were sequenced using Titanium chemistry Roche. The sequencing data were analyzed using mothur v. Briefly, sequence barcodes and primers were removed, sequences were denoised with the mothur translation of the PyroNoise algorithm and trimmed to remove sequences if they contained ambiguous bases, homopolymers greater than 8, or less than total bases. The remaining sequences were aligned to the SILVA bacterial database alignment region start position ; Pruesse et al.
Remaining sequences were preclustered for sequences differing by a single base, and Uchime was used to detect chimeras Edgar et al. Chimeras were removed from the dataset. The classifier database was modified with additional sequences and phylogenetic classification detail for the known FeOB McBeth et al. The datasets from the 16 analyzed samples were normalized by random subsampling to include an equal number of reads each. Each reaction was prepared in triplicate and each plate also contained a standard dilution series of an amplified and quantified target gene, no template controls, and negative and positive control samples.
For the DsrA gene assay, the standard series and positive controls were prepared from amplified Desulfovibrio salexigens DsrA gene, and the negative control was prepared using the estuarine iron mat amplified SSU rRNA gene detailed above.
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We single-cell sorted samples from the BBH site and conducted single-cell genomics analysis of the steel biofilms. The two sample points 9 and 22 days were selected based on expected peak abundances of FeOB populations determined from our qPCR data observations from the GSB site time series. Each steel sample was placed in a 15 ml conical tube, 2 ml of filter sterilized seawater was added, the sample was gently agitated to dislodge flocculent microbial corrosion biofilm material from the coupon, and the biofilm material was transferred to a 2 ml centrifuge tube.
The material was mixed five times with a 16G needle and syringe to break up particulates and microbes from the mineral matrix, and the material was gently homogenized using a vortex mixer. The samples were centrifuged briefly to remove large particles and diluted in filtered seawater. Sequences were aligned and classified using mothur as described for high-throughput amplicon sequencing analyses.
At the GSB saltmarsh site, the sampler was placed on soft sediments typical of a tidal saltmarsh. At the low water point, the sampler was above the water surface. We observed a salt wedge in the stream when the tide was high; higher salinity water inundating the salt marsh at high tide and freshwater flowed out in the stream at low tide.
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As the study progressed, the sample holders accumulated corrosion products and sediment particulates which caused them to retain water even during low tide. At the BBH site, the samples were placed in a protected boat harbor on soft bottom, approximately 0. The maximal tidal range during the study was on the order of 3. The steel coupons developed orange flocculent coatings within the 1st week of each incubation; by the end of each study the coatings were still predominantly orange but also included patches of black or greenish precipitates Figures 1E—G.
Stalk structures indicative of FeOB were observed in microscopy images of the steel corrosion products at timepoints throughout the incubations Figures 1A—D. Microscopy images of stalk structures characteristic of FeOB from field incubations and macroscopic images of corrosion biofilms. Amplicon sequencing was used to understand the community dynamics of populations colonizing steel coupons over time at the brackish site and full strength seawater site. We obtained between 1, and 44, reads for each of the coupon and sediment samples. Two samples from the BBH site one of the two 9-day coupons and the day sediment sample failed in PCR amplification, and so were eliminated from the dataset.
Stress and R 2 values for these ordination plots were A 0. Colored ovals encompass replicate samples where applicable. At the phylum level, the corrosion biofilm and sediment communities in both series were dominated by Proteobacteria and Bacteriodetes Figures 2A,B. The presence of Zetaproteobacteria in the saltmarsh sediments was consistent with small but visible iron mats we observed adjacent to the incubation chamber at the GSB site.
At the BBH site, sequences from the Zetaproteobacteria were present from 9 days onward, but were not observed in the sediments Figures 2D,F. Another notable difference between the sediment and steel biofilm community samples was the abundance of Epsilonproteobacteria. At the GSB site, the predominant genera of Epsilonproteobacteria were related to sulfur-oxidizers Sulfurimonas , Sulfuricurvum , and Sulfurovum.
Relatives of a sulfide-oxidizing Arcobacter species also represented a large portion of the Epsilonproteobacteria in the early GSB biofilm samples, but their abundance decreased over the course of the incubation.
The Deltaproteobacteria on the steel biofilms increased through time in each series and reads from this class were high in the sediment samples, as expected Figures 2C,D. These orders included reads that classified as relatives of the sulfate- and iron-reducing bacteria but in contrast to the GSB data, we did not observe reads classifying as Geobacter or Geopsychrobacter in the BBH site data.
The Alphaproteobacteria reads at both sites were dominated by the Rhodobacteriaceae; generally speaking, Alphaproteobacteria were richer in the GSB site samples in comparison with the BBH site samples.
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Gammaproteobacteria at the GSB site included relatives of methanotrophs, methane-oxidizers, and sulfur oxidizers, and at the BBH site we identified relatives of several known sulfur oxidizers. Reads classified as Shewanellaceae, potential iron-reducers, were identified in both data sets. The amplicon results revealed members of the Bacteroidetes were remarkably abundant at the BBH site. These percentages are much higher than have been seen in other coastal sediments Wang et al. At both sites, the Bacteroidetes were dominated by the class Flavobacteria, which contains a diverse group of aerobic heterotrophs.
The sediment samples at the GSB site had particularly high diversity estimates, exceeding the other samples by nearly an order of magnitude. Figures 3A,B contain the Beta diversity results.
At both field sites we observed a trend in the community structure changes over the course of the experiments. The initial timepoint samples from BBH clustered close to the sediment sample from that site. Time series qPCR results. A Results from GSB site duplicate steel coupon extracts, B results from single stream sediment samples taken from sediment adjacent to the GSB site sampler, C results from BBH site triplicate steel coupon extracts, and D results from single sediment samples taken adjacent to the BBH site sampler. All data is gene copies per ng of DNA in extract.
Error bars represent standard error of replicate samples where applicable; if not visible they fall within the boundaries of the symbol. Estimates of the abundance of Zetaproteobacteria on steel coupons at the GSB site based on qPCR indicated they were already detectable at 3 days, increased up to 10 days, and then declined gradually over the next 30 days. The qPCR data for the GSB site sediment was relatively constant, with Zetaproteobacteria about 1 to 2 orders of magnitude less abundant than SRB, and about 1 to 2 orders of magnitude less abundant than their maximal abundance measured on the coupons at 10 days.
In contrast, at the BBH site the DsrA gene copies started out at about 10 6 gene copies per ng of DNA and decreased gradually over time, with a final concentration around 10 4 gene copies per ng of DNA. Here the Zetaproteobacteria abundance was essentially at or below the assay detection limit at 3 days, but then showed a large increase in abundance by days 6 and 13, followed by fluctuations and a general decline over time. In the BBH site sediments, Zetaproteobacteria were at concentrations at or below the detection limit of the assay throughout the study, and DsrA gene copies were on the order of 10 6 gene copies per ng of DNA.
Single cell sorts from the BBH site were successful and resulted in identifiable sequences from cells 9-day sample and cells day sample. Pie charts show percent of each class of Proteobacteria observed in each sample. All of these SAGs grouped with relatives of the known sulfur-oxidizing genera Sulfurimonas and Arcobacter ; in addition the day 9 SAG library had one Sulfurovum relative. The work presented here shows that mild steel corrosion communities are dynamic over time and there are successional patterns in the microbial communities.
In both the brackish and seawater incubations, members of the Zetaproteobacteria that are presumptive FeOB were enriched on the steel surface, confirming earlier studies that FeOB are an important part of the MIC community Dang et al. At GSB, incubations were done in a locale where there was visible Fe-oxidation, and presumably Fe-reduction, occurring at the sediment interface in proximity to the steel coupons. In this case the Zetaproteobacteria were detectable in the sediments, but were enriched by 1 to 2 orders of magnitude on the coupons.
The incubation site at the fully marine BBH site was on soft bottom sediment with no visible evidence for Fe-cycling i. Nonetheless, the same pattern of rapid colonization of the steel surface by FeOB occurred, and based on qPCR data, the abundance of Zetaproteobacteria on the steel surface reached a similar order of magnitude as for the brackish site at GSB. In both cases, it appeared the Zetaproteobacteria population reached an apex after 10—12 days and then declined. These results show that FeOB are early colonizers of steel surfaces, and are consistent with the findings of Dang et al.
However, as the community develops, their relative abundance decreases, suggesting that the steel surface niche is changing to become a more mature biofilm with anoxic zones that support the growth of strict anaerobes like SRB. This is consistent with previously models for mature steel corrosion communities Hamilton, ; Little and Lee, This study is unique in looking at both brackish and marine environments with initiation of corrosion under fully aerobic conditions, nor are we aware of studies, aside from the work of Dang et al.
These dynamics indicate that the surface niche has not stabilized and that continued physicochemical changes may select for different populations, while at the same time ecological interactions between populations are also continuing to shape and alter the community. A longer time course would be necessary to determine if the MIC community reaches a stable composition. Deltaproteobacteria were expected to be part of the community, since this class includes a known sulfate- and Fe-reducing genera and both of these physiological groups have been previously observed in association with marine MIC communities e.
The relative abundance of Deltaproteobacteria, as assessed by amplicon sequencing, increased with time at both sites. At GSB, copy numbers of DsrA were low at 15 days and then increased over time, this is consistent with formation of a more mature biofilm with anoxic niches that could support sulfate-reduction. At BBH, DsrA copies were relatively abundant initially, on a par with the sediments, and then decreased over time, while sediment numbers stayed quite constant.
We do not have a good explanation for this pattern, and it is inconsistent with the relative increase in reads of Deltaproteobacteria in the amplicon analysis. One possibility is that Fe-reducing Deltaproteobacteria may have been a larger part of the community than SRB at the early timepoints, however, this is not reflected in the classification of the sequencing results.
It has also recently been shown that some Alphaproteobacteria belonging to the Roseobacter clade contain DsrA genes and it is possible they may have also been targeted by the qPCR primers Lenk et al. The presence of Bacteroidetes on the BBH sampler coupons through most of the time course was especially pronounced.
A more detailed assessment of the SSU rRNA amplicon reads for this group indicated the closest known relatives were members of the Flavobacteriales that includes aerobic and facultative heterotrophs that are often saprophytic Krieg et al. The work of Dang et al. The specific role of this group in the MIC community is difficult to ascertain, although it is possible they are taking advantage of primary production by chemolithotrophic Fe- and S-oxidizing bacteria. It is also known that members of the Flavobacteriales are efficient at colonizing surfaces and growing as biofilms Doghri et al.
Another possible explanation for the high abundance of Bacteroidetes at the BBH site is that they are often excellent organic matter decomposers, and the environment was rich in particulate organic matter. More specifically, there was a significant amount of seaweed in the vicinity of the sampler, although it was not in direct contact with the samples.
This seaweed was starting to undergo senescence and degradation in late summer, which would likely promote the growth of saprophytic microbes Thomas, The high number of Epsilonproteobacteria identified in the SAG libraries at both time points was interesting. These represent absolute cell numbers in the sorted sample, which included cells from the loosely adhering portion of the biofilm. The Epsilonproteobacteria were consistently present in the amplicon libraries, but at a lower relative abundance. Members of the Epsilonproteobacteria are not commonly reported as being important members of the MIC community; however, Dang et al.
Our findings are consistent with that report, as the identified genera included Sulfurimonas and Arcobacter , known S-oxidizers Campbell et al. However, it cannot be ruled out that some of these organisms may also be carrying out H 2 -oxidation e. The presence and relative abundance of Zetaproteobacteria SAGs on the 9-day BBH samples in comparison with the samples taken at 22 days was consistent with the general decline in Zetaproteobacteria abundance observed from qPCR over the same timeline.
It is worth noting we did not obtain any Deltaproteobacteria in the SAG libraries. Although it is possible this is bias of selecting a particular population of high nucleic acid cells via flow cytometry sort or bias against either lysis or amplication of this group in the SAG preparation, we believe a more likely explanation may have to do with the structure of the biofilm, and how it was sampled.
The samples for SAG analysis were taken only from a comparatively loose surface layer of the biofilm, where more aerobic members of the community would be expected. For high-throughput amplicon sequencing and qPCR analyses the entire surface layer consisting of the outer, loosely adherent layer, and an inner, more adherent layer was used in order to obtain enough nucleic acid for the analyses.
Thus it is possible the SAG libraries are selecting organisms with more aerobic metabolisms, which is consistent with the relative abundance of Zetaproteobacteria and Epsilonproteobacteria. The population structure of FeOB within the biofilms showed important differences between brackish and fully marine conditions. At GSB, where the salinity fluctuated from between 0 and 21 ppt as a result of tidal influence, representatives of the freshwater FeOB, Gallionellaceae were found in addition to Zetaproteobacteria.
This is consistent with an earlier study that looked at the distribution of FeOB along a salinity gradient and found overlap between these groups in brackish regions McBeth et al. This also implies that freshwater FeOB are part of MIC communities in strictly freshwater habitats, and is consistent with their presence on steel corrosion products in the Great Lakes Hicks, , scale from reclaimed wastewater Jin et al. In fact, our analysis of Zetaproteobacteria SAGs from BBH indicated a very low diversity with only two clones accounting for the abundance of Zetaproteobacteria on the samples.
One explanation for this is that a low overall abundance of Zetaproteobacteria in the coastal ocean at least at this location leads to recruitment of only a few strains that become established on the steel surface and grow to abundance. This observation is supported by the clonal Zetaproteobacteria sequences we were able to amplify in a previous coastal study of corrosion biofilms McBeth et al. Interestingly, basalt coupons incubated in the abyssal plain of the deep ocean are also known to recruit Zetaproteobacteria that presumably gain energy from the reduced iron within the basalt.
These communities are also of low diversity, suggesting a similar mechanism of recruitment Henri et al. In this study, we have characterized the succession of mild steel corrosion communities over 40 days in both nearshore marine and estuarine salt marsh environments. This is the first time a detailed study of corrosion community succession has been conducted over a period of several weeks. We found that iron-oxidizing Zetaproteobacteria and Betaproteobacteria were able to rapidly colonize the steel surfaces over the first 10 days of the incubations; however, their numbers dropped after this initial colonization and as the corrosion community matured on the steel surfaces.
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